Partially double-stranded nucleic acids, methods of making, and use thereof

Abstract

A process is disclosed for generating at least one partially double-stranded target nucleic acid, which contains at least one single-stranded region at a terminal end. The process comprises the steps of (a) providing at least one primer, P1, containing at least one labile nucleotide; (b) combining at least one target nucleic acid sequence with P1 to generate a double-stranded polynucleotide containing at least one labile nucleotide; (c) exposing the double-stranded polynucleotide to conditions that promote single-strand cleavage of the polynucleotide at the site of the at least one labile nucleotide of primer P1; and (d) exposing the cleaved polynucleotide to conditions that promote the dissociation of the cleaved portions of primer P1 from a terminal end. The labile nucleotide may be dUTP, wherein the single-stranded cleavage of the polynucleotide at the site of the labile nucleotide occurs by treatment with uracil N-glycosylase.

Description

FIELD OF THE INVENTION[0001] The present invention provides methods for generating partially double-stranded nucleic acid molecules that contain at least one terminal single-stranded region available for binding to a complementary nucleic acid. Such molecules are useful for a wide variety of applications, including gene expression analysis, and are particularly useful as targets in DNA microarray analysis and related studies. Because these molecules may be generated without performing a strand separation step, they provide a substantial advantage over traditionally amplified target molecules. In combination with a bipartite primer sequence, the present method provides a universal DNA array index system that can detect or determine the amount of any polynucleotide target.BACKGROUND OF THE INVENTION[0002] Tens of thousands of genes, and potential genes, have been identified in the human genome alone. Many times that number are known and suspected in medically and economically critical...

AI Technical Summary

Benefits of technology

[0019] The invention further comprises methods for the synthesis and use of the above polynucleotides. In an exemplary embodiment, an oligonucleotide primer having one or more deoxyuracil bases is used in a polymerase chain reaction ("PCR"), or other polymerase reaction, to synthesize a differentially labile double-stranded molecule. Enzymatic digestion of the deoxyuracil aids in generating single-strand breaks in the end of the double-stranded molecule, to thereby fragment the labile primer. These fragments have a lower melting temperature than the remainder of the molecule and are easily disassociated from it. The resulting molecule is, thus, partially double-stranded but has a single-stranded region generally corresponding to the labile primer. The single-stranded region is then available to hybridize with a complementary target sequence, and may be used to bind, detect, or quantify a target molecule, e.g., in a nucleic acid microarray.

[0021] Consequently, a universal test kit or DNA array comprising the standard sequence probe affixed to a solid support may be used to detect or determine any target, merely by varying the target-specific 3' end of the bipartite primer. Moreover, the standardization afforded by the universal system allows for accurate comparison of target levels irrespective of the time or place of the assay.

Problems solved by technology

However, conventional PCR results in double-stranded nucleic acid products, whereas DNA chip technology is based on the binding of complementary single-stranded regions on the probe and target.

This strand separation step can be problematic and inefficient because the complementary strands tend to rapidly rehybridize to each other before attaching to the microarray.

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